Adenomatous Polyposis Coli Modulates Actin and Microtubule Cytoskeleton at the Immunological Synapse to Tune CTL Functions Marie Juzans, Céline Cuche, Thierry Rose, Marta Mastrogiovanni, Pascal Bochet, Vincenzo Di Bartolo, Andrés Alcover

To cite this version:

Marie Juzans, Céline Cuche, Thierry Rose, Marta Mastrogiovanni, Pascal Bochet, et al.. Adenomatous Polyposis Coli Modulates Actin and Microtubule Cytoskeleton at the Immunological Synapse to Tune CTL Functions. ImmunoHorizons , The American Association of Immunologists, Inc, 2020, 4 (6), pp.363-381. ￿10.4049/immunohorizons.2000044￿. ￿pasteur-02975339￿

HAL Id: pasteur-02975339 https://hal-pasteur.archives-ouvertes.fr/pasteur-02975339 Submitted on 22 Oct 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés.

Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License Adenomatous Polyposis Coli Modulates Actin and

Microtubule Cytoskeleton at the Immunological Synapse to Downloaded from Tune CTL Functions

Marie Juzans, Céline Cuche, Thierry Rose, Marta Mastrogiovanni, Pascal Bochet, Vincenzo Di Bartolo and Andrés Alcover http://www.immunohorizons.org/ ImmunoHorizons 2020, 4 (6) 363-381 doi: https://doi.org/10.4049/immunohorizons.2000044 http://www.immunohorizons.org/content/4/6/363 This information is current as of October 22, 2020.

Supplementary http://www.immunohorizons.org/content/suppl/2020/06/24/4.6.363.DCSupp

Material lemental at Institut Pasteur - CeRIS on October 22, 2020 References This article cites 58 articles, 26 of which you can access for free at: http://www.immunohorizons.org/content/4/6/363.full#ref-list-1 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://www.immunohorizons.org/alerts

ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE

Adaptive Immunity

Adenomatous Polyposis Coli Modulates Actin and Microtubule Cytoskeleton at the Immunological Synapse to Tune

CTL Functions Downloaded from

Marie Juzans,*,† Céline Cuche,* Thierry Rose,‡,1 Marta Mastrogiovanni,*,† Pascal Bochet,‡ Vincenzo Di Bartolo,*,2 and Andrés Alcover*,2 *Unité Biologie Cellulaire des Lymphocytes, Département d’Immunologie, Institut Pasteur, INSERM-U1221, Ligue Nationale contre le Cancer- † ` ‡ Equipe Labellisée Ligue 2018, F-75015 Paris, France; Sorbonne Université, College Doctoral, F-75005 Paris, France; and Bioimage Analysis Unit, http://www.immunohorizons.org/ Department of Cell Biology and Infection, Institut Pasteur, CNRS-UMR3691, F-75015 Paris, France

ABSTRACT Adenomatous polyposis coli (Apc) is a cell polarity regulator and a tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Apc involvement in T lymphocyte functions and antitumor immunity remains poorly understood. Investigating Apc-depleted human CD8 T cells and CD8 T cells from ApcMin/+ mutant mice, we found that Apc regulates actin and microtubule cytoskeleton remodeling at the immunological synapse, controlling synapse morphology and stability and lytic granule dynamics, including targeting and fusion at the synapse. Ultimately, Apc tunes cytotoxic T cell activity, leading to tumor cell killing. Furthermore, at Institut Pasteur - CeRIS on October 22, 2020 Apc modulates early TCR signaling and nuclear translocation of the NFAT transcription factor with mild consequences on the expression of some differentiation markers. In contrast, no differences in the production of effector cytokines were observed. These results, together with our previous findings on Apc function in regulatory T cells, indicate that Apc mutations may cause a dual damage, first unbalancing epithelial cell differentiation and growth driving epithelial neoplasms and, second, impairing T cell–mediated antitumor immunity at several levels. ImmunoHorizons, 2020, 4: 363–381.

Received for publication May 28, 2020. Accepted for publication June 3, 2020. Address correspondence and reprint requests to: Dr. Vincenzo Di Bartolo and Dr. Andrés Alcover, Lymphocyte Cell Biology Unit, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France. E-mail addresses: [email protected] (V.D.B.) and [email protected] (A.A.) ORCIDs: 0000-0001-6085-2473 (M.M.); 0000-0002-5453-947X (V.D.B.); 0000-0002-9507-3450 (A.A.). 1Current address: Lymphocyte Cell Biology Unit, Department of Immunology, Institut Pasteur, Paris, France. 2Equal contribution as senior authors. This work was supported by grants from La Ligue Nationale contre le Cancer, Equipe Labellisée 2018, and institutional grants from the Institut Pasteur and INSERM. M.J. was supported by a Ligue Nationale contre le Cancer Doctoral Fellowship and the Institut Pasteur. M.M. is a scholar of the Pasteur Paris University International Doctoral Program, supported by the Institut Pasteur and the European Union Horizon 2020 Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement 665807 (COFUND-PASTEURDOC). The Unit of Technology and Service Photonic BioImaging core facility is supported by the French National Research Agency France BioImaging (ANR-10–INSB–04, Investments for the Future). M.J. designed, performed, and analyzed experiments, developed experimental models, and wrote the manuscript; C.C. designed, performed, and analyzed experiments, developed experimental models, and provided technical and laboratory management support; T.R. designed, performed, and analyzed experiments, and developed experimental models. M.M. performed and analyzed some experiments and provided technical support and critical reading. P.B. wrote software for rupture force flow experiments. V.D.B. conceived the project, designed and performed experiments, and wrote the manuscript. A.A. conceived the project, designed experiments, and wrote the manuscript. Abbreviations used in this article: Apc, adenomatous polyposis coli; ConA, concanavalin A; CV, chamber volume; 3D, three-dimensional; ICAReB, Clinical Investigation and Access to BioResources; shRNA, short hairpin RNA; siApc, siRNA for Apc depletion; siCtrl, siRNA control; siRNA, small interfering RNA; TIRF, total internal reflection fluorescence; Treg, regulatory T cell. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY-NC 4.0 Unported license. Copyright © 2020 The Authors https://doi.org/10.4049/immunohorizons.2000044 363

ImmunoHorizons is published by The American Association of Immunologists, Inc. 364 ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS ImmunoHorizons

INTRODUCTION particularly, the anti-inflammatory capacity of Tregs, which could contribute to tumor development in patients carrying Apc TCRs recognize peptide Ags associated with MHC molecules on mutations. APCs. Ag interaction induces early TCR signaling, leading to the NFAT is involved in cytotoxic T cell differentiation and T cell polarization toward APCs and the formation of the function (31). Furthermore, microtubule-mediated lytic granule immunological synapse at the T cell Ag-presenting cell interface. polarization and release at cytotoxic T cell synapses is crucial for Immunological synapses are key to control T cell activation efficient tumor cell killing (7). Therefore, we hypothesized that leading to T cell growth, differentiation and cytokine production. Apc deficiency might also affect cytotoxic T cell functions, They also control T cell effector functions such as polarized reducing their capacity to eliminate tumor cells and contributing secretion of cytokines by Th and regulatory T cells (Tregs) and to tumor escape in Apc-dependent polyposis patients. To lytic granules by CTLs (1). investigate this, we studied Apc-silenced human primary CD8 Immunological synapse generation and function depend on the T cells and CD8 T cells from ApcMin/+ mice. These heterozygous Downloaded from orchestrated action of the actin and microtubule cytoskeleton and mutant mice have been largely used as an animal model to of intracellular vesicle traffic that polarize at the T cell side of the investigate the molecular bases of Apc-mediated intestinal immunological synapse. This drives the targeting and dynamic polyposis and carcinoma (19). clustering of T cell Ag receptors and signaling molecules to In this study, we show that Apc is involved in microtubule and optimally control T cell activation (2). Furthermore, the Golgi and actin cytoskeleton remodeling at the immunological synapse of Min/+ lytic granule intracellular trafficreorienttowardthesynapse, CD8 T cells. Furthermore, CD8 T cells from Apc mice http://www.immunohorizons.org/ delivering Th cytokines directly to Ag-presenting B cells (3–5) or displayed reduced T cell Ag receptor-mediated Erk and Akt kinase lytic granules to infected or tumor target cells. Polarized secretion activation and NFAT nuclear translocation. However, mild or no of lytic granules depends on the fine concomitant dynamic effects were observed on some CD8 differentiation markers or remodeling of actin and microtubule cytoskeleton at the immu- production of cytokines. Importantly, synapse stability, lytic nological synapse (6–9). granule dynamics and fusion at the synapse, as well as cytotoxic Cell polarity complexes are key to ensure stable cell polarity activity against tumor cells were reduced in ex vivo–differentiated (10) as well as induced polarization in migrating cells (11). Scribble, CTLs from ApcMin/+ mice or from Apc-silenced human CD8 Dlg1, and PKCz polarity regulators were shown to control T cells. These results reveal a novel role of Apc in modulating lymphocyte migration, immunological synapse formation, and cytotoxic T cell responses, with potential consequences in – T cell activation (12 17). The polarity regulator and tumor antitumor immunity. at Institut Pasteur - CeRIS on October 22, 2020 suppressor adenomatous polyposis coli (Apc) is known for its association with familial adenomatous polyposis, human co- lorectal tumors, and intestinal carcinomas in mice (18–21). Apc MATERIALS AND METHODS interacts with a variety of proteins, including transcription factor regulators as b-catenin, polarity regulators, such as Dlg1 Human cell isolation, small interfering RNA transfection, or Scribble, cytoskeleton regulators, such as Cdc42 or EB1, lentiviral infection, CTL generation, and tumor target nuclear pore and nuclear transport proteins, and apoptosis- or cell culture mitosis-related proteins (22, 23). Human peripheral blood T cells from healthy volunteers were Apc mutations alter intestinal epithelium differentiation and obtained from the French Blood Bank Organization (Etablisse- induce tumor progression in colorectal cancer patients and in ment Français du Sang) or through the Institut Pasteur Biological mouse models (18–22, 24). The role of Apc in immune responses, Resources Core Facility, Clinical Investigation and Access to in particular against tumors, is much less explored. However, BioResources (ICAReB) (NSF 96-900 certified, from sampling to altered intestinal immune homeostasis and control of inflamma- distribution, reference BB-0033-00062/ICAReB platform/Institut tion by Tregs was reported in Apc mutant mice (25–28). We Pasteur, Paris, France/BBMRI AO203/1 distribution/access: 2016, have previously unveiled that Apc underpins various molecular May 19th, [BIORESOURCE]), under CoSImmGEn protocol ap- mechanisms controlling CD4 T cell functions (29). These include proved by the Committee of Protection of Persons, Ile de France-1 microtubule network organization and centrosome polarization (No 2010-dec-12483). Informed consent was obtained from all at the immunological synapse and NFAT-driven cytokine gene donors. activation. Interestingly, Apc regulates NFATc2 nuclear trans- PBMCs were isolated by centrifugation through Ficoll-Hypaque. location upon T cell activation. Furthermore, NFATc2 forms CD8 T cells were isolated from PBMCs using the MACS CD8 microclusters associated with microtubules and needs microtu- T Cell Isolation Kit (Miltenyi Biotec) and maintained in human bules to translocate to the nucleus upon T cell activation. Finally, in CD8 medium: RPMI 1640 plus GlutaMAX-I (Life Technologies) ApcMin/+ mutant mice, lamina propria Tregs display lower capacity supplemented with 10% FBS, 1 mM sodium pyruvate and nonessential to differentiate and produce cytokines, mainly IL-10 (29). This amino acids, 10 mM HEPES, 1% penicillin–streptomycin (v/v). cytokine is central to regulation of intestinal inflammation and To generate CTLs, freshlyisolated CD8 T cells were stimulated adenocarcinoma progression (30). These findings suggested 2 d with coated anti-CD3 (10 mg/ml; UCHT1 produced by that Apc mutations may also affect immune cell functions and, A. Alcover), soluble anti-CD28 (7 mg/ml; Beckman Coulter), and

https://doi.org/10.4049/immunohorizons.2000044 ImmunoHorizons ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS 365 recombinant human IL-2 (100 U/ml; PeproTech) in human CD8 10% FBS, 1 mM sodium pyruvate and nonessential amino acids, medium. Cells were infected for 24 h with lentiviruses coding for 50 mM 2-ME, 10 mM HEPES, and 1% penicillin–streptomycin control or Apc-specific short hairpin RNAs (shRNAs) (10% v/v) (v/v), in the presence of IL-2 (10 ng/ml; Miltenyi Biotec). in human CD8 medium with IL-2, in which FBS was replaced by human serum and then selected for 3 d with puromycin Apc quantification (3.9 mg/ml). The percentage of infected CTLs was 70–85%, as For Apc detection by Western blot, total cell extracts were assessed by GFP expression by FACS. obtained by lysing cells for 5 min on ice in lysis buffer (50 mM Tris For short interfering RNA (siRNA) experiments, the following [pH 7.4], 100 mM NaCl, 0.5% Nonidet P-40, 5 mM EDTA, 5 mM small dsRNA oligonucleotide sequence was used for Apc depletion EGTA, 40 mM 2-ME, 10 mM NaF, 10 mM Na4P2O7,2mM (siRNA for Apc depletion [siApc]): 59-GAGAAUACGUCCACAC orthovanadate, and protease inhibitor mixture). Cell lysates were CUU-39 (GE Healthcare), as we previously described (29). As cleared by spinning at 20,800 3 g for 20 min at 4°C. An equal siRNA control (siCtrl), the sequence used was 59-UAGCGA amount of protein content was measured using the Bio-Rad Downloaded from CUAAACACAUCAA-39 (siGENOME Non-Targeting siRNA #1; Protein Assay (Bio-Rad Laboratories) was loaded in NuPAGE GE Healthcare). A total of 1 3 107 freshly isolated CD8 T cells were 3–8% Tris-Acetate gels (Thermo Fisher Scientific). Proteins were transfected with 1 nmol siCtrl or siApc using the Human T Cell transferred onto nitrocellulose membranes (LI-COR Biosciences) Nucleofector Kit and the program U-14 on an Amaxa Nucleofector for 4 h using a Bio-Rad Mini Trans-Blot system in buffer II (Lonza). Cells were then harvested in human CD8 medium containing 50 mM Tris, 380 mM glycine, 20% ethanol, and 0.1% without penicillin–streptomycin and used 72 h after transfection. SDS. Membranes were saturated with blocking buffer (Rockland http://www.immunohorizons.org/ For shRNA experiments, lentiviruses were produced by Immunochemicals) and incubated with anti-Apc (2 mg/ml; ALi HEK293T cells transfected with the transient calcium phos- 12–28; Abcam) and anti-ZAP70 (50 ng/ml; BD Biosciences) or phate DNA precipitation technique. Cells were transfected with anti-PLCg1 (1/1000; Cell Signaling Technology) overnight at 4°C. pCMV-deltaR8-2 and pCMV-env-VSV, together with a pLKO.1- They were washed and then incubated with specificsecondary puro-CMV-tGFP lentiviral vector expressing or not (as negative Abs conjugated with Alexa Fluor 680 or DyLight 800 (Thermo control) as shRNA–targeting Apc (59-GACTGTCCTTTCACCA Fisher Scientific) for 45 min. Near-infrared fluorescence was imaged TATTT-39) (Sigma-Aldrich). Forty-eight hours later, supernatant and quantified using the Odyssey Classic Near-Infrared Imaging was recovered and concentrated 403 by ultracentrifugation System (LI-COR Biosciences), and the Apc band intensity was (26,000 rpm, 1.5 h, 4°C). Lentiviruses stocks were stored at 280°C. normalized to control ZAP70 or PLCg1band.

The P815 mouse mastocytoma cell line was used as the tumor For Apc detection by retrotranscription quantitative PCR, total at Institut Pasteur - CeRIS on October 22, 2020 target cell. P815 cells were maintained in DMEM supplemented RNA was extracted using the RNeasy Plus Micro Kit (QIAGEN) with 10% FBS and 1% penicillin–streptomycin (v/v). To make following the manufacturer’s instructions. cDNA was obtained stimulatory target cells, P815 were pulsed with anti-human or from 200 ng of total RNAusing an iScript cDNA SynthesisKit(Bio- mouse CD3 Abs, as described below. Rad Laboratories). FastStart Universal SYBR Green PCR Master Mix (Roche) and an ABI PRISM 7900HT Sequence Detection ApcMin/+ and wild-type mice, lymphocyte isolation, and system (Applied Biosystems) were used to quantify gene products. cell culture Quantitative PCR were performed in triplicates. Quantity values Heterozygous C57BL/6J-ApcMin (ApcMin/+) mice and wild-type were calculated by the relative standard curve method and controls were purchased from The Jackson Laboratory. Mice were normalized to the mRNA expression of the RLP13a housekeep- housed and bred under pathogen-free conditions in the Central ing gene. The primer sequences used were as follows: Apc Animal Facility of the Institut Pasteur. The protocols used had forward, 59-CCAACAAGGCTACGCTATGC-39 and reverse, 59- been approved by the Ethical Committee for Animal Experimen- TACATCTGCTCGCCAAGACA-39; RLP13a forward, 59-CATA tation of the Institut Pasteur and by the French Ministry of GGAAGCTGGGAGCAAG-39 and reverse, 59-GCCCTCCAAT Research. Wild-type and ApcMin/+ male and female littermates CAGTCTTCTG-39 were sacrificed at 8–14 wk of age. The age, gender, and number of mice analyzed per type of experiment are depicted in Confocal microscopy, image posttreatment, and Supplemental Table I. Individual mice were analyzed separately. quantitative image analysis We did not observe any significant gender effect in any of the Coverslips were washed with HCl–ethanol70%andcoatedwith experiments performed. Data from male and female mice were poly-L-lysine 0.002% in water (Sigma-Aldrich). For flat pseudo- pooled for each experimental condition. synapse formation, coverslips were further coated with anti-CD3 Spleen and lymph nodes were homogenized through a 70-mm (10 mg/ml; UCHT1; BioLegend) overnight at 4°C. Coverslips were filter. To generate CTLs, CD8 T cells were purified using the washed and blocked for 30 min at 37°C with human CD8 medium. MACS CD8a T Cell Isolation Kit (Miltenyi Biotec) and stimulated Human T cells were plated on coverslips for 5 min at 37°C and 2 d with coated anti-CD3 (5 mg/ml; 145-2C11; eBioscience), soluble fixed with 4% paraformaldehyde for 13 min at room temperature. anti-CD28 (2 mg/ml; eBioscience), and IL-2 (10 ng/ml; Miltenyi For microtubule detection, cells were also incubated 20 min at Biotec). Cells were then cultured for 5–6 d in mouse CD8 medium: 220°C in ice-cold methanol. For CTL–target cell immunological DMEM plus GlutaMAX-I (Life Technologies) supplemented with synapse formation, P815 cells, previously coated with anti-CD3

https://doi.org/10.4049/immunohorizons.2000044 366 ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS ImmunoHorizons

(20 mg/ml; OKT3 produced by A. Alcover) for 45 min at 4°C, then 10 CV of PBS. Bovine fibronectin (10 mg/ml in PBS; Merck- were plated on poly-L-lysine–coated coverslips for 15 min at Sigma) was incubated 1 h at room temperature and rinsed with 20 37°C. Coverslips were carefully washed with PBS, and human CV of PBS. The chamber was equilibrated with DMEM (Life CTLs were added at a 1:1 CTL/target ratio and incubated for 15 Technologies) at 37°C, then loaded with P815 cells for 15 min at min at 37°C. Cells were then fixed with 4% paraformaldehyde 37°C. One CV of anti-CD3 (20 mg/ml final; OKT3 produced by for 13 min at room temperature. Coverslips were washed in PBS A. Alcover) in DMEM was carefully added, and cells were and incubated 1 h in PBS with 1% BSA (v/v) to prevent unspecific incubated for 15 min at 37°C. Unbound cells and Abs were cleared binding. Cells were then incubated 1 h at room temperature with by flowing five CV of RPMI 1640 (37°C; Lonza). One CV of human PBS, 1% BSA, 0.1% Triton X-100 and anti-Apc (1/300; gift of CTLs in RPMI 1640–1% FBS (Lonza) was injected inside the I. Nathke,¨ University of Dundee), anti–b-tubulin (6.6 mg/ml; chamber and incubated for 10 min at 37°C. A flow rate of PBS, Merck Millipore), anti-pericentrin (1/100; Abcam), and anti- increasing from 0 to 38.4 ml/min, was applied through the perforin (10 mg/ml; BD Biosciences). Coverslips were then chamber for 115 s using a syringe pump (SP210iW, World Preci- Downloaded from incubated with the corresponding fluorescent-coupled second- sion Instruments) controlled by a computer and synchronized ary Ab and Texas Red-X Phalloidin (1/100; Invitrogen) for 45 min with image acquisition (three images per s, 1100 3 840 mm) using at room temperature. After three washes in PBS with 1% BSA, an inverted transmission microscope (Axio Observer D1; Zeiss) coverslips were mounted on microscope slides using ProLong with a 103/0.3 numerical aperture objective controlled by Gold Antifade Mountant with DAPI (Life Technologies). MicroManager (34).

Confocal images were acquired with an LSM 700 confocal Image series were analyzed using ImageJ software (32) and the http://www.immunohorizons.org/ microscope (Zeiss) using the Plan Apochromat 633/1.40 numer- plugin Cell Counter. P815 and CTLs were distinguished by their ical aperture objective. Optical confocal sections were acquired size,shape,andnucleus/cellratio.Theflow rate value at the with ZEN software (Zeiss) by intercalating green and red laser cell–cell rupture event was used to compute the dragging force on excitation to minimize channel cross-talk. All analyses were the released cell according to its size (mean diameter of 8 mm), performed using Fiji software (32). For Apc quantification and shape (roughly spherical), and density (mean value 1.20 kg/l). F-actin analysis, optical sections were acquired at 1-mmintervals. Calibration of dragging force was performed from the sedimen- Apc fluorescent intensity was measured on the total cell. tation rate of cells in the chamber (measured PBS density 1.0034 Formation of the actin ring and phalloidin fluorescence intensity kg/l and dynamic viscosity 0.6998 mPa per s at 37°C) (35) and the analyses were performed on one confocal section at the cell– theoretical flow speed versus wall distance according to Poiseuille fi – coverslip contact. F-actin intensity pro les were assessed along a solution to Navier Stokes formalism for a Reynolds number below at Institut Pasteur - CeRIS on October 22, 2020 line drawn across each cell image (as shown in Fig. 2D). Ranking of 10 characterizing a laminar flow. The number of cells loaded in the each cell in a category (presence of F-actin ring plus central chamber and located in the field of view may vary from one clearance, intermediate; low, F-actin ring plus clearance) was experiment to another. Moreover, the CTLs initially retained as decided after observing actin profiles on three to four different conjugates were always lower for Apc-silenced cells, indicating angles. For microtubule pattern and synapses analyses, optical initial adhesion impairment. To give the same weight to each of sections were acquired at 0.2-mm intervals, and images were the four experiments performed on different cell numbers in treated by deconvolution with the Huygens Professional software the overall whisker-boxes, shCtrl (n = 3745) and shApc (n = (Scientific Volume Imaging). Microtubule pattern analysis was 1091) cells released per force interval were scaled to a total performed on projection of four confocal sections at the cell– number of 250 cells per experiment, 1000 cells after adding the coverslip contact, and phenotypes were classified by blinded image four sets. observation by three different investigators. Synapse morphol- ogy and cytotoxic granule localization were analyzed in three- Live-cell total internal reflection fluorescence microscopy dimensional (3D) projections and classified as described for Glass-bottom microwell dishes (MatTek) were washed with microtubule patterns. Centrosome localization was estimated by HCl–ethanol 70%. They were coated with poly-L-lysine 0.002% measuring the distance between the anti-pericentrin staining in water (Sigma-Aldrich) for 30 min at room temperature and then and the CTL plasma membrane at the center area of the with anti-CD3 (10 mg/ml; UCHT1; BioLegend) overnight at 4°C. immunological synapse. Quantification of cytotoxic granules per cell Human CTLs were incubated with LysoTracker Deep Red was obtained using the TrackMate plugin for ImageJ developed by (0.1 mM; Molecular Probes by Life Technologies) for 45 min at J.-Y. Tinevez (Photonic BioImaging, Unit of Technology and Service, 37°C to label cytotoxic granules. Cells were washed and resuspended Institut Pasteur) (33). in human CD8 medium. A total of 1 3 105 cells were dropped in a microwell, and once cells were seeded, images were acquired in Rupture force assay of cell–cell interactions in laminar live-cell total internal reflection fluorescence (TIRF) plan every flow chamber 150 ms for 4 min. TIRF images were acquired with an LSM 780 Glass surface (Rogo-Sampaic) of the flow chamber (Slide I0.1,Ibidi, Elyra PS.1 confocal microscope (Zeiss) using an a Plan Apo 1003/ Germany) was washed using five chamber volumes (CV) of 1.46 numerical aperture oil immersion objective. Images were sulfuric acid (2 M; Merck-Sigma), then five CV of hydrogen analyzed using the TrackMate plugin for ImageJ software (33). A peroxide (33%; Merck-Sigma), and rinsed with 10 CV of water, fluorescence threshold above 23 the mean granule fluorescence

https://doi.org/10.4049/immunohorizons.2000044 ImmunoHorizons ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS 367 was set to select strong fluorescence events more likely closer to Calbiochem). Membranes were then incubated with specific the plasma membrane. secondary Abs conjugated with Alexa Fluor 680 or DyLight 800 (Thermo Fisher Scientific) for 45 min at room temperature. Near- Cytotoxicity assay infrared fluorescence was recorded and quantified using an At day 6–8 after initial stimulation, human or mouse CTLs were Odyssey Classic Near-Infrared scanner (LI-COR Biosciences). resuspended in RPMI 1640 plus GlutaMAX-I (Life Technologies) Band intensity was normalized to control GAPDH. For a pooled 3% FCS, 10 mM HEPES, and mixed at the indicated ratios with analysis of several experiments, normalized intensities were then P815 target cells previously coated with anti-human-CD3 (6 mg/ml; divided by the mean normalized intensity of the same experiment. UCHT1; BioLegend) or anti-mouse-CD3 (6 mg/ml; 145-2C11; eBioscience) for 45 min at 4°C. Mixed CTLs and target cells were Nuclear NFAT detection incubated 4 h at 37°C. The percentage of target cell lysis was Nuclear NFAT was analyzed by Western blot analysis of cells measured using the CytoTox 96 Non-Radioactive Cytotoxicity fractionated for nucleus and cytoplasm separation. Mouse T cells Downloaded from Assay (Promega) following manufacturer’s instructions. Absor- were activated as for analysis of protein phosphorylation, for the bance at 490 nm was measured using a PR2100 Microplate Reader indicated times. Cells were lysed in ice-cold low-salt lysis buffer (Bio-Rad Laboratories). (10 mM KCl, 10 mM HEPES, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM

DTT, 50 mM NaF, 10 mM sodium pyrophosphate [Na4P2O7], and a Degranulation assay protease inhibitor mixture) for 15 min on ice. Detergent Nonidet

At day 6–8 after initial stimulation, human or mouse CTLs were P-40 (Sigma-Aldrich) was added (0.45% v/v final), and cells were http://www.immunohorizons.org/ mixed at a 1:1 ratio with P815 target cells previously coated with the vortexed for 10 s to break cytoplasmic membranes without alter- indicated anti-CD3 concentration (for human: OKT3, produced by ing nuclear membranes. Lysates were centrifuged at 20,800 3 g A. Alcover; for mice: 145-2C11; eBioscience) in presence of anti- for 30 min at 4°C. Supernatants corresponding to cytosolic fractions CD107a-PE (for human: 1/90, clone H4A3; BioLegend; for mice were recovered in ice-cold tubes. Pellets containing nuclei were 2.5 mg/ml, clone 1D4B; BD Biosciences), and incubated for 3 h at washed once in PBS and resuspended in ice-cold high-salt buffer 37°C. Alternatively, CTLs were stimulated for the indicated time (20 mM Tris–HCl [pH 8], 1% SDS, 2 mM EDTA, and protease with coated anti-CD3 (10 mg/ml; clone OKT3 produced by A. Alcover) inhibitor mixture), solubilized by sonication (3 3 10 s at 60% and soluble anti-CD28 (7 mg/ml; Beckman Coulter). Cells were power) using a Vibracell 72434 (Bioblock Scientific), then stained with either anti-human CD8a-allophycocyanin-Cy7 (1/25; centrifuged at 20,800 3 g for 30 min. Supernatants were recovered

BioLegend) or anti-mouse CD8a-PerCP-Cy5.5 (1 mg/ml; BD as nuclear fractions. An equal amount of protein content was at Institut Pasteur - CeRIS on October 22, 2020 Biosciences). Events were acquired on a MACSQuant Analyzer measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories) (Miltenyi Biotec), and analysis was performed using FlowJo 10 was loaded in NuPAGE 3–8% Tris-Acetate gels (Thermo Fisher software (FlowJo). All samples were gated on forward and side Scientific). Protein transfer was performed in a Bio-Rad Mini scatter and for singlets. Trans-Blot system. Membranes were saturated with blocking buffer (Rockland Immunochemicals) and incubated with anti- Analysis of protein phosphorylation NFATc2 (NFAT1) (0.25 mg/ml; BD Biosciences), anti-SLP76 Mouse T cells were incubated with 20 mg/ml of biotin-conjugated (1/1000; Thermo Fisher Scientific) or anti-Lamin B1 (1/2000; anti-CD3 (145-2C11; eBioscience) and anti-CD28 (eBioscience) Abcam) overnight at 4°C. Then, they were incubated with specific Abs for 30 min at 4°C. Cells were washed, resuspend in Opti-MEM secondary Abs conjugated with Alexa Fluor 680 or DyLight 800 plus GlutaMAX-I (Life Technologies), and placed 1 min at 37°C. (Thermo Fisher Scientific) for 45 min. Near-infrared fluorescence Streptavidin (10 mg/ml; Sigma-Aldrich) was added, and cells were was imaged and quantified using the Odyssey Classic scanner as incubated at 37°C for the indicated times. Ice-cold PBS containing described above. NFAT band intensity was normalized to SLP76 2 mM orthovanadate and 0.05% sodium azide was added to stop cytoplasmic control or Lamin B1 nuclear control. For a pooled cell stimulation. Cells were lysed in ice-cold lauryl–b-maltoside analysis of several experiments, normalized intensities were then buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 0.25% lauryl- divided by the mean normalized intensity of the same experiment. b-maltoside,50mMNaF,10mMNa4P2O7, 1 mM EGTA, 2 mM orthovanadate, 1 mM MgCl2, and protease inhibitor mixture) for Mouse T cell differentiation and proliferation assays 10 min on ice. Cell lysates were cleared by spinning at 20,800 3 g Mouse cells from 12-wk-old mice freshly isolated from the spleen for 10 min at 4°C. Equal amounts of protein content measured and lymph nodes were stimulated with concanavalin A (ConA; using the Bio-Rad Protein Assay (Bio-Rad Laboratories) was 5 mg/ml; Sigma-Aldrich) in mouse CD8 medium with IL-2 loaded in NuPAGE 4–12%Bis-Trisgels(ThermoFisherScientific). (10 ng/ml; Miltenyi Biotec) for 2 d and then cultured four addi- Protein transfer was performed using the Trans-Blot Turbo system tional days with IL-2. At day 0, 2, 4, and 6, cells were incubated with (Bio-Rad Laboratories). Membranes were saturated with blocking anti-CD16/32 (10 mg/ml; BioLegend) to block Fc receptors and buffer (Rockland Immunochemicals) and incubated overnight at 4°C then stained with Fixable Viability Stain 450 (25 ng/ml; BD or 2 h at room temperature with anti–phospho-Akt (p-Ser473; 1/ Biosciences), anti-CD3-FITC (5 mg/ml; 145-2C11, BD Biosci- 1000; Cell Signaling Technology), anti–phospho-Erk1/2 (p-Thr202/ ences), anti-CD4-allophycocyanin-Vio770 (1.5 mg/ml; Miltenyi Tyr204; 1/1000; Cell Signaling Technology), anti-GAPDH (6.6 mg/ml; Biotec), anti-CD8-PerCP-Cy5.5 (1 mg/ml; BD Biosciences),

https://doi.org/10.4049/immunohorizons.2000044 368 ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS ImmunoHorizons

ABC Downloaded from http://www.immunohorizons.org/ D at Institut Pasteur - CeRIS on October 22, 2020 E

F

FIGURE 1. Apc is expressed in CD8+ T cells. (A) Expression of Apc in purified primary human CD8 and CD4 T cells. Cell lysates were analyzed by Western blot using anti-Apc and anti-PLCg1 Abs. The band corresponding to Apc (;310 kDa) was quantified with respect to the PLCg1 band. Data are the mean 6 SEM of three donors. (B) Expression of Apc detected by Western blot in resting primary human CD8 T cells transfected with control or Apc siRNA. Apc band intensity was normalized with respect to ZAP70. Data are the mean 6 SEM of two experiments. (C) Expression of Apc detected by quantitative (Continued)

https://doi.org/10.4049/immunohorizons.2000044 ImmunoHorizons ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS 369 anti-CD25-PE-Cy7 (2 mg/ml; BD Biosciences), anti-granzyme RESULTS B-A647 (1/50; BioLegend), anti-CD44-allophycocyanin (2 mg/ml; eBioscience), and anti-CD62L-RPE (2 mg/ml; eBioscience). Apc is expressed in human CD8 T cells Events were acquired on a MACSQuant Analyzer (Miltenyi We first investigated the expression of Apc in primary human CD8 Biotec), and analysis was performed using FlowJo 10 software T cells. Immunoblotting on cell lysates revealed similar levels in (FlowJo). All samples were gated on forward and side scatter for CD4 and CD8 T cells of a protein band consistent with the 311 kDa singlets, and for live cells, samples were stained using Fixable full-length protein previously described (24, 29) (Fig. 1A). This Viability Stain 450 (250 ng/ml; BD Biosciences). band disappeared upon siRNA silencing in CD8 T cells (Fig. 1B). Proliferation was calculated by counting the cells at day 0, 2, 4, Apc depletion was similarly detected by quantitative RT-PCR and 6 upon stimulation, as described above for the differentiation upon siRNA transfection in human resting CD8 T cells or shRNA assays. Cell numbers were referred to the cell counts at day 0. retroviral transduction in differentiated CTLs (Fig. 1C). We next analyzed Apc subcellular localization. Both resting and differen- Downloaded from Detection of cytokine production tiated CD8 T cells showed a punctate pattern of Apc associated IL-2 was removed from CTL culture, and 20 h later, cytokine with microtubules and aligned at the edges of immunological production was assessed by ELISA and flow cytometry as follows: synapses formed on anti-CD3–coated coverslips (arrowheads) for detection by ELISA, 1 3 3.105 human or mouse CTLs were (Fig. 1D, 1E). This Apc pattern was consistent with our findings in restimulated at 37°C for 6 h with PMA (50 ng/ml; Sigma-Aldrich) CD4 T cells (29) and with previous reports in other cell types (36, and calcium ionophore A23187 (500 ng/ml; Sigma-Aldrich) or 37). The specks pattern was strongly diminished in Apc-silenced http://www.immunohorizons.org/ 20 h with coated anti-CD3 (for human, 5 mg/ml, UCHT1; Bio- T cells (Fig. 1F), further supporting the specificity of the Apc Legend; for mice, 145-2C11, 5 mg/ml; eBioscience) and soluble anti- immunofluorescence pattern. CD28 (for human, 5 mg/ml; Beckman Coulter; for mice, 2 mg/ml; eBioscience). Secreted IL-2, TNF-a,andIFN-g levels were Apc regulates microtubule and actin cytoskeleton measured from culture supernatants with specificDuoSetELISA remodeling at the immunological synapse Kits (R&D Systems) following manufacturer’sinstructions. Apc was shown to regulate microtubule network organization in For detection by flow cytometry, 1 3 105 mouse CTLs were polarized migrating cells (38–40)aswellasactinpolymerization restimulated as described above. Two hours after the beginning of (41). Furthermore, Apc coordinates actin polymerization and restimulation, brefeldin A (10 mg/ml; Sigma-Aldrich) was added. microtubules at focal adhesions in migrating cells (42). In both fi Cells were xed with 2% paraformaldehyde for 15 min at room CD4 and CD8 T cells, actin and microtubule cytoskeletons at Institut Pasteur - CeRIS on October 22, 2020 temperature, incubated with anti-CD16/32 (10 mg/ml; Bio- cooperate to build functional immunological synapses (2, 8, 14). Legend), and subsequently stained with anti-IL-2-RPE (4 mg/ml; Indeed, in CD8 T cells, microtubule polarization toward target eBioscience), anti-TNF-a-FITC (1.2 mg/ml; Miltenyi Biotec) and cells combined with actin polymerization, followed by rapid anti-IFN-g-PE-Cy7 (8 mg/ml; BioLegend) in presence of 0.05% clearance from the center of the synapse, appear to be key for saponin. Events were acquired on a MACSQuant Analyzer cytolytic granule release and target cell lysis (6, 9, 43). Importantly, (Miltenyi Biotec), and analysis was performed using FlowJo 10 we previously showed that Apc silencing in CD4 T cells results software (FlowJo). All samples were gated on forward and side in microtubule network disorganization at the immunological scatter for singlets, and for live cells, they were stained using synapse (29). Fixable Viability Stain 450 (250 ng/ml; BD Biosciences). Therefore, we investigated whether Apc controls cytoskeleton remodeling in CD8 T cell immunological synapses. Apc silencing Statistics altered microtubule network organization at the immunological Statistical analyses were carried out using Prism Software pseudosynapses formed by either resting or differentiated CD8 (GraphPad). Error bars in plots represent the mean 6 SEM. The T cells on anti-CD3–coated coverslips. Microtubule patterns were p values are represented as follows: ****p , 0.0001, ***p , 0.001, more frequently radially organized in control cells, reaching the **p , 0.01, *p , 0.05, NS p $ 0.05. The type of test used is synapse periphery and with visible microtubule organizing center. mentioned in each figure legend. In contrast, Apc-silenced cells more often displayed randomly

RT-PCR in resting primary human CD8 T cells transfected with control or Apc siRNA (top) or ex vivo–differentiated CTLs infected with control or Apc shRNA expressing retroviruses (bottom). Data are the mean 6 SEM of three and five experiments, respectively. Significance was determined by Mann–Whitney U test. (D–F) Resting human CD8 T cells (D and F) or ex vivo–differentiated CTLs (E) were spread on anti-CD3–coated coverslips for 5 min to generate flat immunological pseudosynapses. Cells were then fixed and stained with anti–b-tubulin Ab to reveal microtubules and with anti-Apc Ab. Confocal optical sections were acquired at 0.2-mm intervals. Images were posttreated by deconvolution. A 0.8-mm section at the cell–coverslip contact is shown. Scale bar, 5 mm. (D and E) Right panels are zoomed images of the framed cellular areas corresponding to the periphery of the synapse (D1 and E1) and the center of the cell (D2 and E2). Arrowheads point to Apc puncta. (F) Cells were transfected with control or Apc siRNA. Right panel correspond to the quantification of Apc total fluorescent intensity. Significance was determined by Mann–Whitney U test. **p , 0.01, ****p , 0.0001.

https://doi.org/10.4049/immunohorizons.2000044 370 ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS ImmunoHorizons Downloaded from http://www.immunohorizons.org/ at Institut Pasteur - CeRIS on October 22, 2020

FIGURE 2. Apc controls microtubule and actin cytoskeleton remodeling at the immunological synapse. (A, B, E left, and F left) Human resting CD8 T cells were transfected with control or Apc siRNA. (C–E right, F right) Human ex vivo–differentiated CTLs were infected with retroviruses expressing control or Apc shRNA and GFP. Cells were stimulated on anti-CD3–coated coverslips for 5 min to generate flat immunological synapses. Cells were then fixed and stained with anti–b-tubulin Ab to label microtubules (A–C) or with phalloidin to label F-actin (D–F). (A–C) Confocal optical sections were acquired at 0.2-mm intervals. Images were posttreated by deconvolution. (Continued)

https://doi.org/10.4049/immunohorizons.2000044 ImmunoHorizons ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS 371 organized microtubules (Fig. 2A–C). In addition, we observed that cells with an increasing slope of 34 pN/s. CTLs interacting with actin remodeling at the synapse was also impaired (Fig. 2D–F). immobilized P815 were stretched by this dragging force until Actin could accumulate at the synapse, and no differences between rupture of the contact and release of the T cells into the stream control and Apc-silenced cells were observed with regard to the (Fig. 3E; Supplemental Video 1). CTL–P815 cell rupture forces total amount of F-actin at the contact site (Fig. 2E). However, ranged from 0 to 4.5 nN, with half of the values between 0.5 and control cells often formed F-actin rings at the periphery of the 2.5 nN, suggesting a large variety of synapse formation kinetics or synapse, excluding actin from the center (Fig. 2D, left panel, and complexity. Median rupture forces were 1.52 and 0.67 nN for Fig. 2F). In contrast, Apc-silenced cells less frequently displayed control and Apc-silenced cells, respectively, indicating that the this actin pattern, more often forming synapses with a less defined interacting force was decreased by loss of Apc expression (Fig. 3F). actin ring and lower clearance from the center (Fig. 2D, center and Interestingly, cumulative plots of cell–cell rupture events versus right panels, and Fig. 2F). dragging force show linear distributions for control cells and hyperbolic distributions for Apc-silenced cells (Fig. 3G). Of note is Downloaded from Apc conditions CTL centrosome polarization, that, although equal cellular input of shCtrl and shApc cells was immunological synapse shape, symmetry, and stability applied to the chamber, the number of CTLs forming initial Our observations on cytoskeleton remodeling prompted us to conjugates with P815 cells before applying flow pressure was lower investigate a larger number of features of immunological synapses in shApc-treated cells in most experiments (n in Fig. 3F legend). formed between human CTLs and anti-CD3–coated P815 tumor This further reflects the lower capacity of Apc-silenced cells to target cells, a model of Ab-mediated T cell cytotoxicity. Following initially stabilize conjugates with P815 target cells. To give the http://www.immunohorizons.org/ the examples of synapses shown in Fig. 3A, we analyzed the same weight to each of the four experiments performed on following: 1) CTL centrosome polarization, assessed by the different cell numbers in the combined data whisker-boxes, shCtrl distance of the centrosome (arrows) to the center of the synapse; (n = 3745) and shApc (n = 1091) cells released per force interval 2) synapse symmetry, assessed by the relative aligned position of were scaled to a total number of 250 cells per experiment, 1000 the centrosome and the T cell and target cell nuclei; and 3) synapse cells after adding the four sets. shape, assessed by the presence of large membrane extensions Therefore, Apc silencing affects centrosome polarization, (arrowheads). Fig. 3A (left panel) provides an example of a immunological synapse shape and symmetry, and the stability of symmetrical CTL–target cell conjugate, with a polarized centro- CTL–tumor target cell interactions. some (arrow) and no large membrane extensions. The right panel

is an example of an asymmetrical conjugate, with the centrosome Apc controls lytic granule dynamics, targeting, and fusion at Institut Pasteur - CeRIS on October 22, 2020 poorly polarized and displaying large cell extensions (arrowheads) at the immunological synapse and a more irregular shape. We observed that Apc silencing did not Perturbation of actin and microtubule remodeling at the alter the number of conjugates (data not shown) but impaired immunological synapse, T cell centrosome polarization, and the T cell centrosome polarization toward the synapse and T cell stability of CTL–target cell interactions of Apc-silenced cells symmetry and increased the presence of large cell extensions (Fig. (Figs. 2, 3) might result in reduced accessibility of lytic granules to 3B–D). These features may reflect or be the cause of the inability of the immunological synapse, reducing the capacity of CTLs to kill Apc-silenced cells to make stable interactions with target cells. target cells (6, 7, 9). To obtain further insight into the effect of Apc silencing on the To investigate this, we first analyzed lytic granule dynamics at stability of CTL–target cell conjugates, we measured the rupture the immunological synapse. Human control or Apc-silenced CTLs force of cell–cell interactions between CTLs and anti-CD3–coated were labeled with LysoTracker and set on anti-CD3–coated P815 cells immobilized in a microscope laminar flow chamber coverslips to obtain flat immunological synapses. Granule undergoing increasing flow rate. A laminar shear flow rate was dynamics were then followed by live-cell TIRF microscopy. We applied, generating dragging forces from 0 to 5 nN on individual measured the following events occurring at the TIRF plan during

A 0.8-mm section at the cell–coverslip contact is shown. Microtubule network organization patterns at the immunological synapse were ranked by observation of unlabeled images by three independent investigators in two categories: pattern 1 (P1), presence of a radial plane of microtubules, or pattern 2 (P2), nonradial organization. Data are the mean 6 SEM of three siRNA experiments and two shRNA experiments in duplicates. Significance was determined by two-way ANOVA. Scale bar, 10 mm (A) and 5 mm (B and C). (D–F) Confocal optical sections were acquired at 1-mm intervals; a section at the cell–coverslip contact is shown. F-actin organization patterns at the immunological synapse were ranked in three categories depending on the pixel intensity plot across the synapse (lower panels): F-actin ring plus clearance, centrally depleted actin, and accumulation at the periphery of the synapse; intermediate, uneven depletion of F-actin at the center of the synapse; low F-actin ring or clearance, F-actin all across the synapse. Analyses were performed on a 1-mm section at the cell–coverslip contact. Data are the mean 6 SEM of two siRNA experiments and three shRNA experiments in duplicates. Significance was determined by two-way ANOVA. Scale bar, 5 mm. (E and F) Quantification of total F-actin (E) and F-actin phenotype (F) at the immunological synapse in human resting CD8 T cells transfected with siCtrl or siApc (left panels) or in human CTLs expressing shCtrl or shApc (right panels). **p , 0.01, ****p , 0.0001.

https://doi.org/10.4049/immunohorizons.2000044 372 ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS ImmunoHorizons

ACB

D Downloaded from

E http://www.immunohorizons.org/

FG at Institut Pasteur - CeRIS on October 22, 2020

FIGURE 3. Apc controls centrosome polarization and immunological synapse shape, symmetry, and stability. (A) Control or Apc-silenced human ex vivo–differentiated CTLs were incubated with anti-CD3–coated P815 for 15 min. Cells were then fixed and stained with phalloidin (red) to label F-actin and anti-pericentrin Ab to label the centrosome (white, arrows). Control and Apc shRNA-infected cells are GFP+ (green). Confocal optical sections were acquired at 0.2-mm intervals. Images were posttreated by deconvolution. Conjugate 3D reconstructions are shown. Arrows point to centrosomes, and arrowheads point to membrane extensions. Scale bar, 5 mm. (B) Centrosome distance relative to the synapse. Data are the mean 6 SEM of two experiments in duplicates. Significance was determined by unpaired t test. (C and D) Morphological analysis of the synapses. Cell symmetry (C) and the presence of protrusions (A, arrowhead, and D) were analyzed by observation of unlabeled images by three independent investigators. Data are the mean 6 SEM of four experiments in duplicates. Significance was determined by unpaired t test. (E–G) Control or Apc-silenced human CTLs were set to interact with anti-CD3–coated P815 tumor target cells previously adhered to a fibronectin-coated laminar flow chamber for 10 min at 37°C. Then, a laminar flow of PBS increasing from 0to38.4 ml/min was applied through the chamber for 115 s using a computer-driven syringe pump and synchronized with image acquisition (three images per s). (E) Representative images of Supplemental Video 1, showing the sequential steps of CTL detachment from the target cell. CTLs are distinguished from target cells by their larger size, cytoplasm, and light refringency. Top line, shCtrl (original magnification 3250), and bottom line, shApc-treated cells (original magnification 3300). Force in nN corresponding to each image is depicted. (F and G) Quantification of rupture forces of interactions between CTLs and anti-CD3–coated P815 cells. Force was calculated as described in Materials and Methods. (F) Four healthy donors (1–4) treated with shCtrl (n =684,671,1189,1201)orwithshApc(n =576,239, 124, 152) were analyzed. Whiskers represent minimum and maximum values, whereas gray and black or red boxes represent second and third quartile framing the median. Plots on the right show the combined data from the four donors. Significance determined by Mann–Whitney U test. (G) Cumulative plots of cell–cell rupture events versus dragging force combining data from the four donors. For each point, mean 6 SEM are shown. *p , 0.05, **p , 0.01, ****p , 0.0001.

https://doi.org/10.4049/immunohorizons.2000044 ImmunoHorizons ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS 373 Downloaded from http://www.immunohorizons.org/ at Institut Pasteur - CeRIS on October 22, 2020

FIGURE 4. Apc controls lytic granule dynamics, targeting, and fusion at the immunological synapse. (A–E) Control or Apc-silenced human ex vivo–differentiated CTLs were incubated with LysoTracker to label lytic granules as part of the late endosomal-lysosomal compartment. Cells were then set on anti-CD3–coated coverslips to generate flat immunological synapses. LysoTracker+ granules were tracked using live-cell TIRF microscopy, which allows the observer to follow vesicles within 200-nm distance from the immuno- logical synapse plasma membrane and therefore detects lytic granule targeting and fusion events (fluorescence burst, arrowhead), as well as granule dynamics at immunological synapse. TIRF images were acquired every 150 ms during 4 min. Quantitative data are the mean 6 SEM of three experiments. (A) The number of lytic granules detected per synapse was used as a readout of granule-targeting events (see also Supplemental Video 2). (B and C) Granule dynamics were estimated by tracking individual granules (C). Scale bar, 3 mm. Tracking duration, average speed, and displacement length were assessed (B). (D and E) Among the detected lytic granules, some made a fluorescence burst, indicative of fusion with the plasma membrane. The number of bursts was used as a readout of granule fusion events. (see also Supplemental Video 2). (F) Control or Apc- silenced human ex vivo–differentiated CTLs were incubated with anti-CD3–coated P815 for 15 min. Cells were then fixed and stained with phalloidin (red) and anti-perforin (white) to label cytotoxic granules. Control and Apc shRNA-infected cells are GFP+. Confocal (Continued)

https://doi.org/10.4049/immunohorizons.2000044 374 ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS ImmunoHorizons Downloaded from http://www.immunohorizons.org/

FIGURE 5. Apc controls CTL killing of tumor target cells. (A and B) Control or Apc-silenced human ex vivo–differentiated CTLs (A) or ex vivo–differentiated CTLs from wild-type or ApcMin/+ mice (B) were at Institut Pasteur - CeRIS on October 22, 2020 mixed with anti-human or mouse CD3–coated P815 tumor target cells at the depicted effector CTL/target cell ratios and incubated for 4 h. Killing was assessed by a colorimetric assay as described in Materials and Methods. Data are the mean 6 SEM of six shRNA experiments and 12 mice in duplicates. Significance was determined by two-way ANOVA. (C and D) Human (C), or mouse (D) CTLs obtained as in (A) and (B) were incubated with P815 tumor target cells coated with anti-human or anti-mouse CD3 at the depicted concentration. Anti-LAMP1-PE (CD107a) was added to the cells and incubated for 3 h. LAMP1 expression was analyzed by flow cytometry. Representative FACS plots are displayed in left panels and the percentage of cells overexpressing LAMP1 upon target cell incubation is shown in right panels. Data are mean 6 SEM of four shRNA experiments and six mice in duplicates. ***p , 0.001, ****p , 0.0001.

4 min recording: 1) the total number of bright fluorescence spots silenced CTLs (Fig. 4B, 4C). Importantly, Apc-silenced cells as a readout of granules targeted to the synapse; 2) the granule displayed fewer fusion events (Fig. 4D, 4E, arrowheads, and movement by tracking individual fluorescent particles at the Supplemental Video 2). Therefore, loss of Apc expression alters synapse; and 3) the number of granules generating a fluorescence lytic granule dynamics, targeting, and fusion at the immunological burst as a readout of granule fusion with the plasma membrane (6, synapse. 9). Representative images are shown in Fig. 4E and Supplemental We next studied immunological synapses formed between Video 2. Quantification showed that Apc-silenced cells displayed CTLs and anti-CD3–coated P815 tumor cells upon 15 min of reduced targeting events (Fig. 4A). Furthermore, lytic granules in CTL–target cell contact. Subcellular localization of lytic granules Apc-silenced cells remained for longer times at the synapse, and was assessed by anti-perforin Ab staining. Interestingly, control their speed was lower than in control cells (Fig. 4B). However, the cells displayed a low number of randomly distributed lytic granules length of displacement of granules was similar in control and Apc- (Fig. 4F, left panel, and Fig. 4G), whereas Apc-silenced cells

optical sections were acquired at 0.2-mm intervals. Images were posttreated by deconvolution. Conjugate 3D reconstructions are shown. Scale bar, 5 mm. (G) Lytic granule localization and quantification. Cells were ranked in three categories depending on granule patterns by blinded image analyses of three in- dependent investigators. Polarized: granules are grouped and localized close to the immunological synapse; grouped: granules are grouped but far from the synapse; random: granules are randomly distributed throughout the cell. The total number of lytic granules was quantified using the TrackMate pluginonImageJ. Data are mean 6 SEM of three experiments in duplicates. Significance was determined by unpaired t test. *p , 0.05, **p , 0.01, ****p , 0.0001, ns $ 0.05.

https://doi.org/10.4049/immunohorizons.2000044 ImmunoHorizons ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS 375 Downloaded from http://www.immunohorizons.org/ at Institut Pasteur - CeRIS on October 22, 2020

FIGURE 6. Apc modulates early T cell activation and NFAT nuclear translocation. Ex vivo–differentiated CTLs from wild-type or ApcMin/+ mutant mice were stimulated with anti-CD3 and anti-CD28 Abs for the indicated times. Cells were subsequently lysed, and the depicted signaling proteins were analyzed by immunoblotting. (A–C) Quantification of Akt and Erk1/2 phos- phorylation following CTLs stimulation. pAkt and pErk1/2 band intensities were normalized with respect to GAPDH used as loading control. Normalized intensities were then divided by the mean normalized intensity of the same experiment before pooling data from multiple experiments. Data are representative of one experiment (B) or are mean 6 SEM of seven mice (C). Significance was determined by two-way ANOVA. (D–I) Quantification of NFAT nuclear translocation following CTLs stimulation. Cell lysates were lysed, separated into cytoplasmic (D) and nuclear (G) fractions, and analyzed by immunoblotting. NFAT band intensity was normalized with respect to SLP76 or lamin B1, used as loading control, then divided by the mean normalized intensity of the same experiment to normalize between different experiments. Data are representative of one experiment (E and H) or are mean 6 SEM of three mice (F and I). Significance was determined by two-way ANOVA. *p , 0.05, ****p , 0.0001. displayed a higher number of granules that were more grouped and however, they are less efficiently targeted and fused to the synapse polarizedtowardthesynapse(Fig.4F,rightpanel,andFig.4G). plasma membrane. As a consequence, a higher number of lytic These data suggest that in Apc-silenced cells lytic granules can granules is retained in CTLs during their interaction with target group close to the centrosome and reorient toward the synapse; cells.

https://doi.org/10.4049/immunohorizons.2000044 376 ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS ImmunoHorizons

FIGURE 7. ApcMin/+ mice do not have defects in T cell numbers in vivo.

Cells from wild-type and ApcMin/+ mice at 8, 10, 12, or 14 weeks of age were extracted from spleen and lymph nodes and counted. Numbers of Downloaded from CD3+ (A), CD3+CD4+ (B), and CD3+CD8+ (C) were calculated after flow cytometry analyses of cells stained with anti-CD3, anti-CD4, and anti-CD8 Abs. Each dot represents a mouse. Horizontal bars are the mean 6 SEM. Significance was determined by two-way ANOVA. ****p , 0.0001.

Apc regulates CTL killing of tumor target cells respond to anti-CD3 plus anti-CD28 stimulation. ApcMin/+ CTLs

We next asked whether the defects in cytotoxic granule dynamics exhibited a mild but significantly reduced capacity to activate Erk http://www.immunohorizons.org/ observed in Apc-silenced cells could impair their capacity to kill and Akt serine-threonine kinases as assessed by the phosphory- tumor target cells. To this end, we used conventional longer lation of regulatory residues of these kinases (Fig. 6A–C). In incubation assays to measure degranulation and tumor target cell contrast, the upstream protein tyrosine kinase ZAP70 was more killing. Both human Apc-silenced CTLs and mouse ApcMin/+ CTLs variably activated, and no significant differences between control were less efficient in killing anti-CD3–coated P815 tumor target and ApcMin/+ cells could be appreciated (data not shown). cells, as assessed by a lactate dehydrogenase–release colorimetric Furthermore, NFATc2 nuclear translocation in response to assay (Fig. 5A, 5B). However, no significant differences were anti-CD3 plus anti-CD28 stimulation was reduced in ApcMin/+ observed in the capacity of these human and mouse CTLs to CTLs (Fig. 6D–I). degranulate, as assessed by measuring cell surface expression of

the LAMP1 luminal epitope CD107a upon CTL contact with anti- Apc defects mildly alter ex vivo differentiation of CD8 at Institut Pasteur - CeRIS on October 22, 2020 CD3–coated P815 tumor target cells for 3 h (44) (Fig. 5C, 5D). T cells To investigate the apparent contradiction between a less efficient Wethen investigated whether Apc mutationwas leading to defects lytic granule fusion at the immunological synapse of Apc-silenced in long-term T cell survival affecting T cell numbers in vivo. To this cells and no alteration in LAMP1 expression, we stimulated the cells end, T cells from the spleen and lymph nodes were phenotyped under the same conditions used for granule fusion detection, and we and countedjust upon the mice’seuthanasia.ApcMin/+ mice did not shortened the kinetics. No significant changes in LAMP1 expression show evident defects because they presented similar T cell between control and Apc-silenced CTLs were observed under these numbers at 8 wk of age and even higher T cell numbers at 10, 12, experimental conditions either (Supplemental Fig. 1). This di- and 14 wk of age (Fig. 7). This T cell number increase coincided chotomy has been reported by others (45) and may account for with the increasing development of intestinal adenomatous polyps LAMP1+ vesicle release in an unpolarized manner in response to in ApcMin/+ mice. calcium signals despite a less efficient lytic granule delivery at Next, we analyzed whether Apc defects could be associated the CTL–target cell interface and reduced killing. with impaired CD8 T cell proliferation and differentiation upon in Altogether, the above described data indicate that Apc is vitro T cell stimulation. Splenocytes and lymph node cells were necessary for efficient microtubule and actin cytoskeleton stimulated with ConA and IL-2, and the proliferation and remodeling conditioning, lytic granule targeting and fusion at expression of the activation/differentiation molecules CD25, the immunological synapse, and ultimately, efficient killing of granzyme B, CD44, and CD62L was assessed at 2, 4, and tumor target cells. 6 d upon activation. Expression of CD44 and CD62L adhesion molecules in CD8 T cells from ApcMin/+ mice appeared mildly Apc modulates T cell signaling and NFAT affected, mainly at day 2, and granzyme B was affected at day 6. nuclear translocation CD62L and granzyme B expression tended to be lower, but the We had shown in CD4 T cells that the polarity regulators Dlg1 and high variability among the animals made differences between Apc were involved in microtubule network organization at wild-type and ApcMin/+ statistically NS (Fig. 8A–D,8G,8H).In the immunological synapse, conditioning NFAT transcriptional contrast, no differences were observed in CD25 expression (Fig. activation (14, 29, 46). We therefore investigated whether Apc 8E, 8F). Interestingly, differences in CD44 and CD62L expression modulation of cytoskeleton remodeling could affect CD8 T cell were more readily observed in CD4 T cells, with more pronounced activation. To this end, we analyzed the capacity of ex vivo– and longer effects, and the level and kinetics of CD62L expression differentiated CTLs from control and ApcMin/+ mutant mice to in CD4 T cells were different from in CD8 T cells (Supplemental

https://doi.org/10.4049/immunohorizons.2000044 ImmunoHorizons ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS 377 Downloaded from http://www.immunohorizons.org/ at Institut Pasteur - CeRIS on October 22, 2020

FIGURE 8. Apc defects mildly affect CD8 T cell differentiation ex vivo. Wild-type and ApcMin/+ mouse cells from spleen and lymph nodes were stimulated for 2 days with ConA, then cultured in the presence of IL-2. CD8 T cell differentiation was assessed at the depicted day after stimulation, quantifying by flow cytometry the expression of the following activation- differentiation markers: CD44, CD62L, CD25, and granzyme B (GzmB). (A, C, E, and G) Flow cytometry profiles of a representative experiment showing CD44, CD62L, and CD25 cell surface expression and intracellular granzyme B, gating on CD3+CD8+ population. (B, D, F, and H) Quan- tification of the expression of CD44, CD62L, CD25, and granzyme B at the indicated days. Each dot corresponds to one mouse (mean 6 SEM; significance was determined by unpaired t test). *p , 0.05, **p , 0.01.

Fig. 2A–F). However, no significant differences in the proliferative CTLs and ApcMin/+ CTLs to produce effector cytokines. Despite capacity were found within the same experimental setting the reduced capacity of ApcMin/+ CTLs to induce NFAT nuclear (Supplemental Fig. 2G–I). translocation, we did not observe significant differences in the These findings show that CD8 T cells from ApcMin/+ mice production of IL-2, IFN-g, or TNF-a by differentiated CTLs present mildly reduced early T cell activation events, such as Erk restimulated with anti-CD3 plus anti-CD28, either by intracellular and Akt activation, and NFAT nuclear translocation as well as mild staining and FACS analysis or by ELISA of cell culture changes in CD44, CD62L, and granzyme proteins expressed supernatants (Fig. 9A, 9B). Similar results were found when during differentiation ex vivo. assessing cytokine production in cell culture supernatants of control and Apc-silenced human CTLs by ELISA (Fig. 9C). Apc defects do not alter ex vivo CTL cytokine production These findings indicate that Apc defects in both human and Because NFAT drives the transcription of a variety of T cell mouse CTLs do not affect their capacity to produce effector CD8 cytokines, we investigated the capacity of Apc-silenced human cytokines under our ex vivo stimulation conditions.

https://doi.org/10.4049/immunohorizons.2000044 378 ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS ImmunoHorizons Downloaded from http://www.immunohorizons.org/ at Institut Pasteur - CeRIS on October 22, 2020

FIGURE 9. Apc defects do not alter CTL cytokine production. (A and B) Ex vivo–differentiated CTLs from wild-type or ApcMin/+ mice were stimulated with anti-CD3 and anti-CD28 Abs for 20 h or PMA and ionomycin for 6 h. The production of TNF-a, IFN-g, and IL-2 was analyzed by intracellular staining and FACS (A) or ELISA (B). Percentage of cytokine-positive cells was quantified by FACS. Data are mean 6 SEM of 12 mice (A). Cytokine secretion was assessed by ELISA. One representative experiment with four mice is shown out of three experiments (B). (C) Control or Apc-silenced human ex vivo–differentiated CTLs were stimulated with anti-CD3 and anti-CD28 Abs for 20 h or PMA and ionomycin for 6 h. Cytokine production was detected by ELISA. Data are mean 6 SEM of three experiments.

DISCUSSION consistent with an impact on actin cytoskeleton dynamics (47), synapse stability, shape, and symmetry were also altered in Apc- Altogether, the data shown in this study unveil a novel role of deficient cells. Apc in cytotoxic T cell effector functions. Apc-silenced human Apc regulation of microtubule organization in CD8 T cells is peripheral blood CD8 T cells allowed us to reveal the importance consistent with the described role of Apc as a polarity regulator in of Apc in cytoskeleton remodeling at the immunological synapse. other cell systems (23). Apc performs this function in association Both microtubules and actin reorganization appeared affected. with other regulators of cell polarity, including Cdc42, Scribble, Thus, the radial organization of microtubules and centrosome Par6, PKCz, and Dlg1, as shown, for instance, in migrating polarization were perturbed, and F-actin ring formation at the astrocytes (40, 48). Furthermore, we have shown before that Dlg1 periphery of the synapse and F-actin exclusion from the center of and Apc control microtubule patterns, centrosome polarization, the synapse were impaired in Apc-defective T cells. Additionally, and signaling microcluster dynamics at the immunological synapse

https://doi.org/10.4049/immunohorizons.2000044 ImmunoHorizons ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS 379 of CD4 T cells (14, 29). Although less extensively studied, Apc may and lytic granules to the target cells, lytic granule distance to the also regulate actin polymerization in synergy with its partner, the immune synapse was higher than that of healthy donor cells. formin family protein mDia (41). In addition, Apc regulates actin- Finally, WAS CTLs displayed impaired synaptic ring formation, microtubule cross-talk at focal adhesions of migrating cells (42). distorted less symmetric CTL–target cell synapses with lower Interestingly, molecular partners of Apc, such as Cdc42 (40) and stability, and delayed lethal hit (45, 54). However, WAS CTLs formins (41), were shown to control actin remodeling and displayed defects in cytokine production, contrary to the Apc- centrosome polarization at the immunological synapse (49, 50). defective CTLs described in this study (45). Therefore, Apc may collaborate with these cytoskeleton regulators Although significant, the differences in tumor cell killing to collectively reorganize both cytoskeleton components at the activity between Apc-defective CTLs and control cells were CTL immunological synapse, thus ensuring their interplay, which modest. This may be due to the fact that ApcMin/+ mice are is crucial for properly tuning T cell functions. heterozygous (homozygous mutation is embryonically lethal) Concomitant centrosome polarization and F-actin exclusion (55) and may express residual levels of Apc protein. Likewise, Downloaded from from the center of the immunological synapse has been shown to shRNA silencing in human primary CTLs is not fully efficient. be key for lytic granule targeting and fusion at the synapse and Hence, remaining Apc protein could account for the observed efficient cytotoxic function against infected or tumor target cells function. Furthermore, other polarity regulators acting together (6,7,9,43).Therefore,impairmentofbothmicrotubuleorgani- with Apc may also compensate Apc defects. Finally, the T cell zation and centrosome polarization, together with F-actin re- activation conditions we used for CTL generation in vitro may organization at the synapse in Apc-defective CTLs, may account compensate, in part, for cytotoxicity defects because of Apc by http://www.immunohorizons.org/ for their altered lytic granule dynamics, targeting, and fusion at the increasing the expression of other proteins. This effect was synapse observed by live-cell TIRF microscopy. Apc-silenced cells described for some forms of familial hemophagocytic lympho- could still concentrate lytic granules in the centrosomal area and histiocytosis because of syntaxin 11 or Munc 18-2 mutations, in polarize them to a certain extent toward the immunological which the addition of IL-2 used to induce T cell proliferation in synapse, suggesting that there is not a complete failure in granule vitro was partially compensating for the loss of effector function transportonmicrotubules.Granulesmaybeabletomovetoward (56, 57). Importantly, subtle differences in CTL cytotoxicity the centrosome and microtubules minus end, a movement using in vitro assays may be predictive of more serious cytotoxic mediated by dynein motors. In contrast, granules do not properly defects in vivo, leading to poor immune responses (58). Therefore, reach the immunological synapse plasma membrane. Disorgani- the small differences in cytotoxicity we observed could have

zation of microtubule network at the immunological synapse likely consequences in long-term antitumor immunity in patients. at Institut Pasteur - CeRIS on October 22, 2020 prevents microtubules from reaching the plasma membrane and, We have previously shown in CD4 T cells that Apc silencing as a consequence, precludes centrosome closing up to the synapse, altered NFAT nuclear translocation and reduced NFAT-driven which facilitates lytic granule docking and fusion (7). Kinesin- transcriptional activation, leading to impaired IL-2 gene expres- mediated transport to the microtubules plus end may also help sion. Furthermore, CD4 T cells from ApcMin/+ mice had reduced granules to reach the synapse plasma membrane (51, 52). As a likely capacity to produce IL-2, IL-4, and IFN-g.Finally,Tregsinthese consequence of inefficient lytic granule dynamics, targeting, and mutant mice showed impaired differentiation and production of fusion, Apc-defective CTLs killed tumor target cells less efficiently the anti-inflammatory cytokine IL-10 (29). We show in this study than control cells. However, no difference in degranulation as that CTLs from ApcMin/+ mice also had an impaired capacity to assessed by increase in LAMP1 (CD107a) cell surface expression translocate NFATc2 to the nucleus. Interestingly, NFATc2 formed was detected. The apparent contradiction between the live-cell microclusters associated with microtubules in CD8 T cells (data TIRF experiments and LAMP1 expression may be due to the not shown), as we previously showed in CD4 T cells (29). The total distinct sensitivities of the two assays to discriminate differences. number of CD3+ lymphocytes, CD3+CD4+ or CD3+CD8+ was not In particular, the fact that the LAMP1 vesicular compartment is reduced in ApcMin/+ mice but even increased starting from 10 wk of larger than the lytic granule compartment and may fuse in a age and stayed enhanced at 12 and 14 wk of age, at which time signs nonpolarized manner in response to normal calcium influx in of polyposis become evident. This suggests that long-term survival Apc-silenced cells (29) could explain this dichotomy of findings. in vivo of CD4 or CD8 lymphocytes is not impaired, but T cell Of note, lack of direct correlation between degranulation and stimulation due to intestinal inflammation at the appearance of cytotoxicity has been reported in other experimental setups (45, polyposis may increase T cell numbers. Interestingly, the analysis 53). of the expression of several differentiation markers (e.g., CD25, Interestingly, our findings share a number of features with granzyme B, CD44, and CD62L) did not show activation of freshly those previously reported in CTLs from Wiscott–Aldrich Syn- isolated T cells from ApcMin/+ mice. In addition, ex vivo activation drome patients, who carry mutations in the gene encoding the with ConA and IL-2 revealed onlymildly altered CD44 and CD62L WAS protein (WASP), a key regulator of actin cytoskeleton expression at day 2 and of granzyme B at day 6 in ApcMin/+ mice. dynamics. Thus, in vitro–derived CTLs from WAS patients had a Interestingly, under the same stimulation conditions, the differ- lower capacity to kill tumor target cells, whereas they display ences in expression of CD44 and CD62L between ApcMin/+ and equivalent degranulation as assessed by LAMP1 expression. control T cells were more pronounced and significant in the CD4 Furthermore, although WAS CTLs could reorient their centrosome T cell population. This was not accompanied by differences in the

https://doi.org/10.4049/immunohorizons.2000044 380 ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS ImmunoHorizons proliferative capacity of CD4 and CD8 T cells under the same 5. Depoil, D., R. Zaru, M. Guiraud, A. Chauveau, J. Harriague, G. Bismuth, stimulatory conditions. This is, in part, in contrast with our previous C. Utzny, S. Muller,¨ and S. Valitutti. 2005. Immunological synapses are versatile structures enabling selective T cell polarization. Immunity 22: data on lamina propria CD4 T cells that displayed impaired – ff 185 194. proliferation capacity at suboptimal stimulation (29). The di er- 6. Ritter, A. T., Y. Asano, J. C. Stinchcombe, N. M. Dieckmann, B. C. Chen, ences may be due to the distinct tissue origin of T cells, lamina C. Gawden-Bone, S. van Engelenburg, W. Legant, L. Gao, M. W. propria in our previous publication and spleen and lymph nodes Davidson, et al. 2015. Actin depletion initiates events leading to granule in this study, or they may be due to the different stimulatory secretion at the immunological synapse. Immunity 42: 864–876. conditions used, different doses of CD3/CD28 on lamina propria 7. Stinchcombe, J. C., E. Majorovits, G. Bossi, S. Fuller, and G. M. Griffiths. 2006. Centrosome polarization delivers secretory granules cells in our previous work versus ConA in this study. In addition, to the immunological synapse. [Published erratum appears in 2006 Min/+ Apc-silenced human and Apc mouse ex vivo–differentiated Nature 444: 236.] Nature 443: 462–465. CTLs produced an equal amount of IL-2, TNF-a, and IFN-g after 8. Dieckmann, N. M., G. L. Frazer, Y. Asano, J. C. Stinchcombe, and restimulation with CD3/CD28 Abs. Therefore, in comparison G. M. Griffiths. 2016. The cytotoxic T lymphocyte immune synapse at Downloaded from with our previous work (29), CD8 T cells appear to be less a glance. J. Cell Sci. 129: 2881–2886. 9. Ritter, A. T., S. M. Kapnick, S. Murugesan, P. L. Schwartzberg, sensitive to Apc defects than CD4 T cells when performing their ffi ff G. M. Gri ths, and J. Lippincott-Schwartz. 2017. Cortical actin re- activation programs leading to di erentiation and production of covery at the immunological synapse leads to termination of lytic Min/+ cytokines. Furthermore, the intestinal environment in Apc granule secretion in cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. mice could also influence these responses. USA 114: E6585–E6594.

Altogether, the data presented in this study show that Apc is 10. Rodriguez-Boulan, E., and I. G. Macara. 2014. Organization and ex- http://www.immunohorizons.org/ involved in the regulation of CTL effector functions that drive ecution of the epithelial polarity programme. Nat. Rev. Mol. Cell Biol. 15: 225–242. target cell killing. Hence, Apc defects impair both actin and 11. Elric, J., and S. Etienne-Manneville. 2014. Centrosome positioning in po- microtubule cytoskeleton remodeling at the immunological larized cells: common themes and variations. Exp. Cell Res. 328: 240–248. synapse, centrosome polarization and lytic granule dynamics, 12.Ludford-Menting,M.J.,J.Oliaro,F.Sacirbegovic,E.T.Cheah,N.Pedersen, targeting, and fusion at the synapse. However, CD8 T cell S. J. Thomas, A. Pasam, R. Iazzolino, L. E. Dow, N. J. Waterhouse, et al. differentiation and cytokine production appear much less de- 2005. A network of PDZ-containing proteins regulates T cell polarity and pendent on Apc compared with what has been observed in CD4 morphology during migration and immunological synapse formation. 22: 737–748. fi Immunity T cells (29). Thus, Apc defects may not signi cantly alter the 13. Real, E., S. Faure, E. Donnadieu, and J. Delon. 2007. Cutting edge: acquisition of effector functions in CD8 T cells but impair the CTL atypical PKCs regulate T lymphocyte polarity and scanning behavior. – capacities to kill tumor cells and could therefore have an impact in J. Immunol. 179: 5649 5652. at Institut Pasteur - CeRIS on October 22, 2020 long-term antitumor immune responses in familial adenomatous 14. Lasserre, R., S. Charrin, C. Cuche, A. Danckaert, M. I. Thoulouze, F. de polyposis patients, allowing easier tumor escape. Chaumont,T.Duong,N.Perrault,N.Varin-Blank,J.C.Olivo-Marin,etal. 2010. Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse. EMBO J. 29: 2301–2314. DISCLOSURES 15. Bertrand, F., M. Esquerré, A. E. Petit, M. Rodrigues, S. Duchez, J. Delon, and S. Valitutti. 2010. Activation of the ancestral polarity regulator protein kinase C zeta at the immunological synapse drives The authors have no financial conflicts of interest. polarization of Th cell secretory machinery toward APCs. J. Immunol. 185: 2887–2894. 16. Xavier, R., S. Rabizadeh, K. Ishiguro, N. Andre, J. B. Ortiz, H. Wachtel, ACKNOWLEDGMENTS D. G. Morris, M. Lopez-Ilasaca, A. C. Shaw, W. Swat, and B. Seed. 2004. Discs large (Dlg1) complexes in lymphocyte activation. J. Cell We thank Drs. F. Sepulveda and D. Scott-Algara for scientific advice, J.Y. Biol. 166: 173–178. Tinevez, A. Salles, J. Fernandes and the Unit of Technology and Service 17. Round, J. L., L. A. Humphries, T. Tomassian, P. Mittelstadt, Photonic BioImaging facility, Institut Pasteur for technical support and the M. Zhang, and M. C. Miceli. 2007. Scaffold protein Dlgh1 coordinates ICAReB Biological Resources core facility team, Institut Pasteur for alternative p38 kinase activation, directing T cell receptor signals providing primary T cell samples. We are grateful to I. Nathke¨ for Abs. toward NFAT but not NF-kappaB transcription factors. Nat. Immu- nol. 8: 154–161. 18. McCartney, B. M., and I. S. Nathke.¨ 2008. Cell regulation by the Apc REFERENCES protein Apc as master regulator of epithelia. Curr. Opin. Cell Biol. 20: 186–193. 1. Huse, M., E. J. Quann, and M. M. Davis. 2008. Shouts, whispers and the 19. Zeineldin, M., and K. L. Neufeld. 2013. More than two decades of Apc kiss of death: directional secretion in T cells. Nat. Immunol. 9: 1105–1111. modeling in rodents. Biochim. Biophys. Acta 1836: 80–89. 2. Soares, H., R. Lasserre, and A. Alcover. 2013. Orchestrating cyto- 20. Su, L. K., K. W. Kinzler, B. Vogelstein, A. C. Preisinger, A. R. Moser, skeleton and intracellular vesicle traffic to build functional immuno- C. Luongo, K. A. Gould, and W. F. Dove. 1992. Multiple intestinal logical synapses. Immunol. Rev. 256: 118–132. neoplasia caused by a mutation in the murine homolog of the APC 3. Kupfer, A., and G. Dennert. 1984. Reorientation of the microtubule- gene. Science 256: 668–670. organizing center and the Golgi apparatus in cloned cytotoxic lymphocytes 21. Moser, A. R., H. C. Pitot, and W. F. Dove. 1990. A dominant mutation triggered by binding to lysable target cells. J. Immunol. 133: 2762–2766. that predisposes to multiple intestinal neoplasia in the mouse. Science 4. Kupfer, A., T. R. Mosmann, and H. Kupfer. 1991. Polarized expression 247: 322–324. of cytokines in cell conjugates of helper T cells and splenic B cells. 22.Nelson,S.,andI.S.Nathke.¨ 2013. Interactions and functions of the ade- Proc. Natl. Acad. Sci. USA 88: 775–779. nomatous polyposis coli (APC) protein at a glance. J. Cell Sci. 126: 873–877.

https://doi.org/10.4049/immunohorizons.2000044 ImmunoHorizons ADENOMATOUS POLYPOSIS COLI TUNES CYTOTOXIC T CELL FUNCTIONS 381

23. Etienne-Manneville, S. 2009. APC in cell migration. Adv. Exp. Med. 42. Juanes, M. A., H. Bouguenina, J. A. Eskin, R. Jaiswal, A. Badache, and Biol. 656: 30–40. B. L. Goode. 2017. Adenomatous polyposis coli nucleates actin as- 24. Béroud, C., and T. Soussi. 1996. APC gene: database of germline and somatic sembly to drive cell migration and microtubule-induced focal adhe- mutations in human tumors and cell lines. Nucleic Acids Res. 24: 121–124. sion turnover. J. Cell Biol. 216: 2859–2875. 25. Gounaris, E., N. R. Blatner, K. Dennis, F. Magnusson, M. F. Gurish, T. B. 43. Le Floc’h, A., Y. Tanaka, N. S. Bantilan, G. Voisinne, G. Altan-Bonnet, Strom, P. Beckhove, F. Gounari, and K. Khazaie. 2009. T-regulatory Y. Fukui, and M. Huse. 2013. Annular PIP3 accumulation controls cells shift from a protective anti-inflammatory to a cancer-promoting actin architecture and modulates cytotoxicity at the immunological proinflammatory phenotype in polyposis. Cancer Res. 69: 5490–5497. synapse. [Published erratum appears in 2017 J. Exp. Med. 214: 1203.] 26. Chae, W. J., and A. L. Bothwell. 2015. Spontaneous intestinal tu- J. Exp. Med. 210: 2721–2737. morigenesis in Apc (/Min+) mice requires altered T cell development 44. Bryceson, Y. T., C. Fauriat, J. M. Nunes, S. M. Wood, N. K. Bjorkstr¨ om,¨ with IL-17a. J. Immunol. Res. 2015: 860106. E. O. Long, and H. G. Ljunggren. 2010. Functional analysis of human 27. Akeus, P., V. Langenes, A. von Mentzer, U. Yrlid, A.˚ Sjoling,¨ P. Saksena, NK cells by flow cytometry. Methods Mol. Biol. 612: 335–352. S. Raghavan, and M. Quiding-Jarbrink.¨ 2014. Altered chemokine pro- 45. De Meester, J., R. Calvez, S. Valitutti, and L. Dupré. 2010. The duction and accumulation of regulatory T cells in intestinal adenomas Wiskott-Aldrich syndrome protein regulates CTL cytotoxicity and is of APC(Min/+) mice. Cancer Immunol. Immunother. 63: 807–819. required for efficient killing of B cell lymphoma targets. J. Leukoc. Downloaded from 28. Tanner, S. M., J. G. Daft, S. A. Hill, C. A. Martin, and R. G. Lorenz. Biol. 88: 1031–1040. 2016. Altered T-cell balance in lymphoid organs of a mouse model of 46. Lasserre, R., and A. Alcover. 2010. Cytoskeletal cross-talk in the control colorectal cancer. J. Histochem. Cytochem. 64: 753–767. of T cell antigen receptor signaling. FEBS Lett. 584: 4845–4850. 29. Aguera-Gonz¨ alez,´ S., O. T. Burton, E. Vazquez-Ch´ avez,´ C. Cuche, 47. Bouchet, J., I. Del Rı´o-Iniguez,~ R. Lasserre, S. Aguera-Gonzalez,¨ F. Herit, J. Bouchet, R. Lasserre, I. Del Rı´o-Iniguez,~ V. Di Bartolo, and C. Cuche, A. Danckaert, M. W. McCaffrey, V. Di Bartolo, and A. Alcover. A. Alcover. 2017. Adenomatous polyposis coli defines treg differenti- 2016. Rac1-Rab11-FIP3 regulatory hub coordinates vesicle trafficwith http://www.immunohorizons.org/ ation and anti-inflammatory function through microtubule-mediated actin remodeling and T-cell activation. EMBO J. 35: 1160–1174. NFAT localization. Cell Rep. 21: 181–194. 48. Osmani, N., N. Vitale, J. P. Borg, and S. Etienne-Manneville. 2006. 30. Rubtsov, Y. P., J. P. Rasmussen, E. Y. Chi, J. Fontenot, L. Castelli, Scrib controls Cdc42 localization and activity to promote cell polar- X. Ye, P. Treuting, L. Siewe, A. Roers, W. R. Henderson, Jr., et al. ization during astrocyte migration. Curr. Biol. 16: 2395–2405. 2008. Regulatory T cell-derived interleukin-10 limits inflammation at 49. Gomez, T. S., K. Kumar, R. B. Medeiros, Y. Shimizu, P. J. Leibson, and environmental interfaces. Immunity 28: 546–558. D. D. Billadeau. 2007. Formins regulate the actin-related protein 2/3 31. Martinez, G. J., R. M. Pereira, T. Aij¨ o,¨ E. Y. Kim, F. Marangoni, M. E. complex-independent polarization of the centrosome to the immu- Pipkin, S. Togher, V. Heissmeyer, Y. C. Zhang, S. Crotty, et al. 2015. nological synapse. Immunity 26: 177–190. The transcription factor NFAT promotes exhaustion of activated 50. Stowers, L., D. Yelon, L. J. Berg, and J. Chant. 1995. Regulation of the CD8+ T cells. Immunity 42: 265–278. polarization of T cells toward antigen-presenting cells by Ras-related 32. Schindelin, J., I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, GTPase CDC42. Proc. Natl. Acad. Sci. USA 92: 5027–5031. T.Pietzsch,S.Preibisch,C.Rueden,S.Saalfeld,B.Schmid,etal.2012.Fiji:an 51. Burkhardt, J. K., J. M. McIlvain, Jr., M. P. Sheetz, and Y. Argon. 1993. open-source platform for biological-image analysis. Nat. Methods 9: 676–682. Lytic granules from cytotoxic T cells exhibit kinesin-dependent mo- at Institut Pasteur - CeRIS on October 22, 2020 33. Tinevez, J. Y., N. Perry, J. Schindelin, G. M. Hoopes, G. D. Reynolds, tility on microtubules in vitro. J. Cell Sci. 104: 151–162. E. Laplantine, S. Y. Bednarek, S. L. Shorte, and K. W. Eliceiri. 2017. 52. Kurowska, M., N. Goudin, N. T. Nehme, M. Court, J. Garin, A. Fischer, TrackMate: an open and extensible platform for single-particle G. de Saint Basile, and G. Ménasché. 2012. Terminal transport of lytic tracking. Methods 115: 80–90. granules to the immune synapse is mediated by the kinesin-1/Slp3/ 34. Edelstein, A. D., M. A. Tsuchida, N. Amodaj, H. Pinkard, R. D. Vale, Rab27a complex. Blood 119: 3879–3889. and N. Stuurman. 2014. Advanced methods of microscope control 53. Wolint, P., M. R. Betts, R. A. Koup, and A. Oxenius. 2004. Immediate using mManager software. J. Biol. Methods 1: e10. cytotoxicity but not degranulation distinguishes effector and memory 35. Kamsma, D., P. Bochet, F. Oswald, N. Alblas, S. Goyard, G. J. L. Wuite, subsets of CD8+ T cells. J. Exp. Med. 199: 925–936. E. J. G. Peterman, and T. Rose. 2018. Single-cell acoustic force 54. Houmadi, R., D. Guipouy, J. Rey-Barroso, Z. Vasconcelos, J. Cornet, spectroscopy: resolving kinetics and strength of T cell adhesion to M. Manghi, N. Destainville, S. Valitutti, S. Allart, and L. Dupré. 2018. fibronectin. Cell Rep. 24: 3008–3016. The wiskott-aldrich syndrome protein contributes to the assembly of 36. Nathke,¨ I. S., C. L. Adams, P. Polakis, J. H. Sellin, and W. J. Nelson. the LFA-1 nanocluster belt at the lytic synapse. Cell Rep. 22: 979–991. 1996. The adenomatous polyposis coli tumor suppressor protein lo- 55. Moser, A. R., A. R. Shoemaker, C. S. Connelly, L. Clipson, K. A. Gould, calizes to plasma membrane sites involved in active cell migration. C. Luongo, W. F. Dove, P. H. Siggers, and R. L. Gardner. 1995. Ho- J. Cell Biol. 134: 165–179. mozygosity for the Min allele of Apc results in disruption of mouse 37. Li, Z., K. Kroboth, I. P. Newton, and I. S. Nathke.¨ 2008. Novel self- development prior to gastrulation. Dev. Dyn. 203: 422–433. association of the APC molecule affects APC clusters and cell mi- 56. Hackmann, Y., S. C. Graham, S. Ehl, S. Honing,¨ K. Lehmberg, gration. J. Cell Sci. 121: 1916–1925. M. Arico,` D. J. Owen, and G. M. Griffiths. 2013. Syntaxin binding 38. Etienne-Manneville, S. 2013. Microtubules in cell migration. Annu. mechanism and disease-causing mutations in Munc18-2. Proc. Natl. Rev. Cell Dev. Biol. 29: 471–499. Acad. Sci. USA 110: E4482–E4491. 39. Etienne-Manneville, S., and A. Hall.2003.Cdc42regulatesGSK-3betaand 57. Bryceson, Y. T., E. Rudd, C. Zheng, J. Edner, D. Ma, S. M. Wood, adenomatous polyposis coli to control cell polarity. Nature 421: 753–756. A. G. Bechensteen, J. J. Boelens, T. Celkan, R. A. Farah, et al. 2007. 40. Etienne-Manneville, S., J. B. Manneville, S. Nicholls, M. A. Ferenczi, Defective cytotoxic lymphocyte degranulation in syntaxin-11 deficient and A. Hall. 2005. Cdc42 and Par6-PKCzeta regulate the spatially familial hemophagocytic lymphohistiocytosis 4 (FHL4) patients. localized association of Dlg1 and APC to control cell polarization. Blood 110: 1906–1915. J. Cell Biol. 170: 895–901. 58. Jessen, B., A. Maul-Pavicic, H. Ufheil, T. Vraetz, A. Enders, 41. Okada, K., F. Bartolini, A. M. Deaconescu, J. B. Moseley, Z. Dogic, K. Lehmberg, A. Langler,¨ U. Gross-Wieltsch, A. Bay, Z. Kaya, et al. N. Grigorieff, G. G. Gundersen, and B. L. Goode. 2010. Adenomatous 2011. Subtle differences in CTL cytotoxicity determine susceptibility polyposis coli protein nucleates actin assembly and synergizes with to hemophagocytic lymphohistiocytosis in mice and humans with the formin mDia1. J. Cell Biol. 189: 1087–1096. Chediak-Higashi syndrome. Blood 118: 4620–4629.

https://doi.org/10.4049/immunohorizons.2000044